A number of proteins referred to in the art as bone morphogenetic proteins (BMPs) have recently been identified which are able to induce bone or cartilage formation when implanted into mammals. For example, Wang et at. in U.S. Pat. No. 5,013,649, incorporated herein by reference, describe the DNA sequences encoding bovine and human bone morphogenetic proteins 2A (now bone morphogenetic protein-2) and 2B (now bone morphogenetic protein 4); the corresponding proteins encoded by those DNA sequences, and processes for recombinant production of the BMP-2A (now BMP-2) and BMP-2B (now BMP-4) proteins. Wozney et al., in U.S. Pat. No. 5,106,748, incorporated herein by reference, describe the DNA and amino acid sequences of bovine and human bone morphogenetic protein-5 (BMP-5), along with processes for recombinant production of the BMP-5 proteins. In U.S. Pat. No. 5,187,076, incorporated herein by reference, Wozney et at. disclose DNA sequences, amino acid sequences, and process for recombinant production of human and bovine bone morphogenetic protein-6 (BMP.-6). DNA and amino acid sequences encoding bone morphogenetic protein-7 (BMP-7, sometimes referred to as OP-1 ) and processes for recombinant production of BMP-7 are described in Rosen, et at., U.S. Pat. No. 5,141,905, incorporated herein by reference. DNA sequences encoding BMP-8 are disclosed in PCT publication WO91/18098. DNA sequences encoding BMP-9 are disclosed in PCT publication WO93/00432. These references are herein incorporated by reference. These proteins are expected to have broad medical applicability in treatment of bone and cartilage injuries and disorders in mammals. In order to fulfill the expected medical need for these bone morphogenetic proteins, large quantities of biologically active protein will be needed.
Recombinant production of the bone morphogenetic proteins is possible both in eukaryotic and prokaryotic cell culture systems. A common occurrence in recombinant production of heterologous proteins in prokaryotic cells, such as bacteria, is the formation of insoluble intracellular precipitates known as inclusion bodies. While the bacteria are generally able to transcribe and to translate DNA sequences encoding heterologous proteins correctly, these prokaryotic cells are unable to fold some heterologous proteins sufficiently correctly to allow for their production in a soluble form. This is particularly true of prokaryotic expression of proteins of eukaryotic origin, such as the bone morphogenetic proteins. Formation of incorrectly folded heterologous proteins has to some extent limited the commercial utility of bacterial fermentation to produce recombinant mammalian proteins. When produced in bacteria, the recombinant bone morphogenetic proteins are often similarly found in inclusion bodies in an aggregated, biologically inactive form.
Several methods for obtaining correctly folded heterologous proteins from bacterial inclusion bodies are known. These methods generally involve solubilizing the protein from the inclusion bodies, then denaturing the protein completely using a chaotropic agent. When cysteine residues are present in the primary amino acid sequence of the protein, it is often necessary to accomplish the refolding in an environment which allows correct formation of disulfide bonds (a redox system). General methods of refolding are disclosed in Kohno, Meth. Enzym., 185:187-195 (1990).
EP 0433225 describes a method for refolding transforming growth factor .beta. T6F-.beta.)-like proteins which employs, in addition to a chaotropic agent and a redox system, a solubilizing agent in the form of a detergent. EP 0433225 predicts that the methods disclosed therein are generally applicable for refolding "TGF-.beta.-like proteins", based on the degree of homology between members of the TGF-.beta. family. However, the present inventors have found that the methods disclosed in EP 0433225 produce undesirably low yields of correctly folded, biologically active dimefie protein when applied to bacterially produced BMP-4, BMP-5, BMP-6, or BMP-7 for unknown reasons.